Apocynin, a cardiotonic constituent of the rhizomes of Apocyanum cannabium [Finnemore Horace, J. Chem. Soc. 93 (1908) 1513-1520] and A. androsaemifolium [Naves Yves-Rene, Helv Chim Acta 32 (1949) 1351] and essential oils of the rhizomes of Iris species have been reported to be responsible for choleretic activity of Picrorhiza kurroa [Basu, K., Dasgupta B., Bhattacharya, S. K. and Debnath, P. K. Curr Sci, 40 (1971) 603]. The constituent has been synthesized and pharmacologically evaluated [Suri, O. P., Bindra, R. S., Satti, N. K. and Khajuria, R. K. Indian Journal of Chemistry, 26B (1987), p 587-88]. Apocynin has also been evaluated for antioxidant and free radical scavenging activities.
Roots of Picrorhiza kurroa are used therapeutically in traditional medicine of almost all Asian countries to treat a manifold of conditions of illness including liver, lung & spleen ailments [Rajaram, D. (1976) Bomb. Hosp. Journal, 18, 66-69; Pandey, G. S. (1979) Indian Materia Medica, Chaukhamba Sanskrit Sausthan, Varanasi pp 70-71; Langar, J. G., Gupta, O. P. and Atal, C. K. (1981), Ind. J. Pharm. 13, 98-99; Handa, S. S., Sharma, A. and Chakraborty, K. K. (1986), Fitotherapia, 58, 307-351; Ansari, R. R., Kapoor, N. K., Kulshreshta, D. K., Mehta, H., Mehrotra, B. N., Patnaik, G. K. and Sharma, S. K. (1988), Indian J. Med. Plants, 87, 401-404] and inflammatory disorders [Nadkarni, K. M. (1954), Indian Materia Medica, vol. 1, Popular Book Depot, Bombay pp. 25-27, 619-622, 634-651, 953-955, 1220-1221, 1252-53; Dey, A. C. (1981), Indian Medicinal Plants used in Ayurvedic Preparations, Bishan Singh & Mahendra Pal Singh, Dehra Dun, India pp 81-82; Jayaweera, D. M. A. (1982) Medicinal Plants used in Ceylon, The National Science Council of Sri Lanka, Colombo, Sri Lanka, part 5, pp 76]. Hepatoprotective, Immunostimulant & Immunorestorative formulations based on P. Kurroa chemical constituents, mainly iridoid glycosides, have been developed at CDRI Lucknow & RRL Jammu [450/ DEL/89 & 845/ DEL/92].
Literature survey revealed that earlier reports showed the presence of trans-tetracos-15-enoic acid in Jojoba oil ex. Simmondsia chinensis seeds (0.62-1.11%) and cis isomer of the acid is reported in fatty acids of the seed oil of Microula sikkimensis (1.2% [Wang Huiying, Yu Xuefian, Yi Yuanfen and Ding Jingkai Yunnan Zhiwu-Yajiu 1989, 11 (1), 60-4 (Ch.), L. Jing Jingmin, Wang Jingping, Yu Fenglan. Zhiwu Xuebao, 1989, 31 (1), 50-3 (Ch.) These reports do not mention isolation of the constituent and the content estimation based on GLC data.
Indigofera tinctoria has been in use in indigenous system of medicine in epilepsy, nervous disorders & bronchitis [Wealth of India, vol. 5. (Council of Scientific & Industrial Research, New Delhi) 182, (1959)]. The plant is also used as ointment in sores, old ulcers and haemorrhoids [R. N. Chopra, S. L. Nayar and I. C. Chopra, Glossary of Indian Medicinal Plants, 141 (1956)]. The leaves of the plants have been used in liver ailments [Nadkarni, K. M., Indian Materia Medica, vol. 1 (Popular Book Depot, Bombay, 680 (1954)]. Extract of the leaves of the plant has exhibited marked hepatoprotective effect against CCl4 induced hepatic injury in rabbits, rats and mice at Regional Research Laboratory (RRL) Jammu. [Anand, K. K., Chand Dewan and Ghatak, B. J. Ray, Indian J. Exp. Biol., 17, 685 (1979); Anand, K. K., Chand Dewan, Ghatak, B. J. Ray and Arya, R. K., Indian J. Expl. Biol., 19, 298 (1981)].
Recent study in RRL Jammu for hepatoprotective effect of the plant extract and further bioactivity-guided fractionation has resulted in identification of trans-tetracos-15-enoic acid as the active principle. The constituent has been synthesized and observed to possess dose related hepatoprotective effect against galactosamine, paracetamol and CCl4 as hepatotoxins using commercially available silymarin as reference material.
Activity of the formulation being described in the invention is not exactly equal to the sum of the activities of the two individual constituents and activity enhancement does not occur simply due to the mixing of the two compounds. This has been verified by mixing the formulation with RLJ-NE-299A, a standardized mixture of iridoid glycosides from Picrorhiza kurroa possessing hepatoprotective, immunostimulant and immunorestorative effects [Indian patent no. 178866].
The mixture of Apocynin, trans-tetracos-15-enoic acid prepared in many other proportions by weight have not shown any enhancement in hepatoprotective action and in some experiments the biological activity of the mixture is much less than the individual constituents.